Structure of a membrane protein in a lipid membrane: cryo-EM study of the HCN channel Abstract Membrane proteins are involved in various critical biological processes, and account for 70% of all known pharmacological targets. However, only about 200 unique membrane protein structures have been identified. The lack of membrane protein structures hinders the understanding of biological reactions at a molecular level and the development of membrane protein drug discovery. This gap is mainly due to the difficulty in forming crystals for X-ray and electron crystallography. We have developed a novel method, called "random spherically constrained" (RSC) single-particle electron cryomicroscopy (cryo-EM). I propose to establish a platform based on the RSC method to determine membrane protein structures in different conformational states to a resolution of 0.4-0.5 nm. First, the RSC method will be applied to study the hyperpolarization- activated cyclic nucleotide-gated (HCN) channel in both open and closed states. Then both experimental and computational methods will be developed to increase the resolution to near- atomic dimension. HCN channels are widely expressed throughout the nervous system and in the heart. They regulate cardiac and neuronal firing rates ("pacemaker" activity), and contribute to several important neuronal processes. The determined structures will provide insights into the gating mechanism of HCN channels, and will potentially facilitate the development of new therapeutic agents to treat diseases in broad areas, such as cardiac arrhythmia. The discoveries will also be applicable to other voltage-gated and ligand-gated channels. This new platform does not rely on crystallization. It preserves protein integrity and activities, and enables access to biologically interesting states that may not be accessible by other methods. This method opens up a new way to study many important membrane proteins, and enables researchers to choose their targets based on the biological importance. PUBLIC HEALTH RELEVANCE: Structure of a membrane protein in a lipid membrane: cryo-EM study of the HCN channel Project Narrative Although membrane proteins account for 70% of all known pharmacological targets, the lack of membrane protein structures hinders the understanding of biological reactions at a molecular level and hinders drug discovery efforts. The proposed method can be, in principle, applied to study the structure of any membrane protein in any functional state. The application of the proposed method to an ion channel, the HCN channel, will enable the understanding of the rhythmic firing ("pacemaker") activity in the brain and heart, and will potentially facilitate the development of new therapeutic agents to treat diseases in broad areas, such as cardiac arrhythmia.